Xanthan gum is an exopolysaccharide (“EPS” hereafter) produced by the phytopathogenic bacterium Xanthomonas campestris (Ielpi et al., 1981, FEBS Lett. 130:253-256). The biosynthesis of this extracellular polysaccharide in X campestris is directed by a cluster of 12 genes, gumB-gumM (Becker et al., 1998, Appl Microbiol Biotechnol, 50:145-152; Vojnov et al., 1998, Microbiology 144; 487-493). It is composed of polymerized pentasaccharide repeating units which are assembled by the sequential addition of glucose-1-phosphate, glucose, mannose, glucuronic acid, and mannose on a polyprenol phosphate carrier (Ielpi et al., 1993, J. Bacteriol. 175:2490-2500). The pentasaccharide repeating unit is also O-acetylated and pyruvylated to various degrees (Ielpi et al., 1993, J. Bacteriol. 175:2490-2500).
Transformation with a gene cluster carrying gumB and gumC restored xanthan production in deficient mutants, suggesting that gumB and gumC are both involved in the translocation of xanthan across the bacterial membrane (Vojnov et al., 1998, Microbiology 144: 487-493).
Two gene groups are involved in xanthan biosynthesis: the genes xpsIII, xps IV and xps VI, which are responsible for synthesis of UDP-glucose, UDP-glucuronic acid and GDP-mannose, respectively (Harding et al., J. Bacteriol 169: 2854-2861 (1987); Harding et al., J. Gen Micobiol 139: 447-457 (1993); Hötte et al., 1999; Köplin et al. J. Bacteriol 174: 191-199 (1992), 1992) and the genes gum or xpsI, which encode the enzymes involved in the polymerization of the pentasaccharide linked to polyprenol phosphate and secretion of the EPS to the extracellular medium (Ielpi et al., 1992; supra Ielpi et al., 1993 supra).
The X. campestris gum genes are grouped in an operon (GUM operon) of about 16 Kb in length, containing 12 genes designated gumB, C, D, E, F, G, H, I, J, K, L and M. The gumD gene encodes the enzyme glucosyltranferase I, responsible for the transference of the glucose-1-phosphate of UDP-glucose to the polyprenol phosphate carrier to form the lipid-monosaccharide. GumM encodes the enzyme glucosyltranferase II, which catalyzes the addition of the second glucose residue to form the lipid-disaccharide. GumH encodes the enzyme glucosyltranferase III, which catalyzes the addition of the first mannose residue to form the lipid-trisaccharide. GumK encodes the enzyme glucosyltranferase IV, which catalyzes the addition of a glucuronic acid residue to form the lipid-tetrasaccharide. GumI encodes the enzyme glucosyltranferase V, which catalyzes the addition of a second mannose residue to form the lipid-pentasaccharide. GumF encodes the enzyme acetyltransferase I which catalyzes acetylation of the internal mannose residue. GumG encodes the enzyme acetyltransferase II which catalyzes the acetylation of the external mannose residue. Finally, gumL encodes the enzyme pyruvate ketal transferase which catalyzes pyruvylation of the external mannose residue. GumB, C and E are involved in the polymerization and secretion of the EPS through the bacterial membrane while gumJ is thought to be involved in a parallel stage of polymerization and secretion of the EPS (Ielpi et al., 1992 supra; Ielpi et al., 1993 supra).
Xanthan has wide commercial application as a viscosifier of aqueous solutions. It is used in both the food and non-food industries and also as a stabilizer of suspensions, emulsions, and foams. Mutant Xanthomonas strains defective in the xanthan biosynthesis pathway have been shown to synthesize and polymerize xanthans with variant structures, similar rheological properties, and different viscosities (Hassler and Doherty, 1990, Biotechnical Prog 6:182-187). Acetylation and pyruvylation can affect the viscometric properties of xanthan. The presence of pyruvate increases viscosity, whereas acetate decreases viscosity (Hassler and Doherty, supra). The elimination of sugar residues from xanthan side chains also affects viscosity. As compared to wild-type xanthan, polymers lacking the terminal mannose (polytetramers) are poor viscosifiers. In contrast, polymers lacking both the terminal mannose and glucuronic acid residues (polytrimers) are superior viscosifiers (Hassler and Doherty, supra). A nonacetylated and an acetylated tetramer, both lacking the side-chain terminal mannose residue and in the first case lacking an acetate group on an internal mannose residue showed higher viscosity (in the first case) and lower viscosity (in the second case) when compared to a wild-type bacteria (Levy et al., 1996, Biopolymers 38:251-272). A major conformational difference between the higher and lower viscosities is the increased amount of open helical backbone and the increase in the side-chain flexibility high viscosity (Levy et al., 1996, Biopolymers 38:251-272).
Xanthan has also been used as a drug stabilizer, to improve drug absorption, to delay drug release and can also be used in controlled-release formulations (Talukdar et al., 1996, J Pharm Sci 185:537-540). In combination with xanthan gum, alpha-cyclodextrin reduced the first-pass metabolism of morphine in the rectal mucosa and by the liver, and was shown to improve the apparent rectal bioavailability of the opioid about 4 fold (Kondo et al., 1996, Biol Pharm Bull 19:280-286). Xanthan-alginate has been used for encapsulation of enzymes. The xanthan-alginate spheres showed 75% of maximum urease activity even after 20 repeated uses under optimal conditions (Elgin, 1995, Biomaterials 16:1157-1161).
Sustained-release hydrogel suppositories prepared with water-soluble dietary fibers, xanthan gum and locust bean gum have been shown to sustain drug release for a much longer time than commercial suppositories. The mean residence time was higher, without a decrease in the area under the plasma concentration vs. time curve. Histopathological studies showed good biological safety of the hydrogel suppositories to the rectal mucosa. These results suggested that IMC hydrogel suppositories prepared with xanthan gum and locust bean gum were a practical rectal preparation having prolonged action and reduced side effects (Watanabe et al., 1993, Bio Pharm Bull 16:391-394).
The apparent release rate of prednisolone from hydrogels prepared with xanthan decreased with increasing gum concentration, suggestion that the diffusion of drug molecules was mainly controlled by the density of the three-dimensional network structure in the matrix. These results indicated that drug release could be controlled not only by the density of the network structure but also by the microscopic viscosity of the hydrogels (Watanabe et al., 1992, Chem Pharm Bull 40:459-462).
A neomycin-furazolidone-xanthan complex has been shown to increase the antimicrobial activity of the drug (Dumitriu et al., 1993, J. Biomater 7:256-276).
Xanthan gum has also been shown to alleviate diabetes mellitus. Administration of xanthan gum lowered fasting and postload serum glucose and reduced fasting levels of total plasma cholesterol in diabetic subjects. Xanthan gum also tended to lower fasting and postload levels of gastrin and gastric inhibitory polypeptide (GIP) and fasting levels of total and VLDL triglyceride and cholesterol in VLDL and LDL fractions. Subjects reported a sense of fullness after consuming xanthan muffins but no severe digestive symptoms (Osilesi et al., 1985, Am J Clin Nutr 42:597-603).
Several factors have been associated with pathogenicity of phytopathogenic bacteria, including production of extracellular enzymes, production of exopolysaccharides, production of toxins and phytohormones along with factors mediating specific plant pathogen interactions (Agrios, 1988, Plant Pathology, 3rd edition, Academic Press; Alippi, 1992, Agronomie 12:115-122). The role of EPSs in the pathogenicity of several bacteria is well known. They are associated with (i) cell death due to occlusion of xylem vessels, (ii) mucoid lesions and (iii) helping plant/pathogen interaction and bacterial growth in the plant tissues.
Several groups have investigated the role of xanthan in Xanthomonas pathogenicity. Although it was believed for a long time that xanthan gum is key to pathogenicity, many authors did not succeed in demonstrating a direct link between this EPS and pathogenicity (Kamoun and Kado, 1990, J. Bacteriol 172:5165-5172). Recently, it has been demonstrated that deletion of gumD (Chou et al., 1997, Biochem Biophys Res. Commun 233:265-269) or alterations in the later stages of xanthan biosynthesis (Katzen et al., 1998, J. Bacteriol 180:1607-1617) reduced virulence in causing black rot in broccoli and decreased the aggressiveness of Xanthomonas against plants. Although the symptoms of X. fastidiosa infection of orange trees are well known, e.g., leaf clorosis, nutritional deficiency symptoms, reduced fruit size, etc. (De Negri, 1990, Com. Tec. No. 82, Ext. Rural, Cats, Compinas; Malavolta et al., 1990, Cordeiropolis, v. 11, n. 1, p. 15-18), the mechanism by which the bacteria causes the disease is unknown. Bacterial infection depends on insect vectors such as Oncometopia facialis, Acrogonia terminalis and Dilobopterus costalimai (Gravena, et al., 1997, “Os vetores de Xylella fastidiosa In: Clorose Variegada do Citros”). These insects, when feeding on xylem nutrients, introduce the bacteria into the vessel where bacteria grows fastidiously (Lopes et al., 1996, “Xylella fastidiosa,” Fitopathologin Brasileira, Brasilia v. 21, Suplemento p. 343; Roberto et al., 1996, Fitopatologia Brasileira, Brasilia v. 21, n. 4, p. 517-18). The EPS synthesized by the XfGUM operon, which is a feature of the invention, might be directly involved in the pathogenicity causing Citrus Variegated Clorosis in orange trees and other diseases in plants, such as coffee and grape plants.